Tobacco addiction is the leading cause of preventable death and is predicted to contribute to more than 8 million deaths worldwide per year by 2030. Despite the impact of this addiction on human health, smoking cessation is a challenge for many, at least in part because addiction is a complex, multifactorial disease with few pharmacological treatments available. It has been shown that extending the half-life of nicotine through decreased metabolism alters human smoking behavior, resulting in fewer cigarettes smoked per day, decreased smoking intensity, and improved smoking cessation rates. Our preliminary results indicate that cinnamic aldehyde, a natural product from cinnamon used extensively in the food industry, is an irreversible inhibitor of the human nicotine metabolizing enzyme, cytochrome P450 2A6 (CYP2A6), which is responsible for metabolizing 70-90% of a nicotine dose. With three specific aims, this project will investigate cinnamic aldehyde as a prototype for the design of new orally administered tobacco cessation agents, with a unique mechanism of action that will improve smoking cessation rates with limited risk for toxicity. Using a combination of fluorescent assays, mass spectrometry, and absorption spectroscopy, Specific Aim 1 evaluates the selectivity, potency, and mechanism for inhibition of human CYP2A6 by cinnamic aldehdye in human liver microsomes, cytosol, and recombinant human enzymes. This analysis will reveal details about the type of structural changes necessary to generate more potent and selective inhibitors. Using molecular modeling, fluorescent assays, and spectral binding studies, Specific Aim 2 evaluates structural analogs of cinnamic aldehyde for their capacity to selectively bind to and inhibit human CYP2A6. The findings from this aim will focus on identifying agents for further investigation as viable drugs. In Specific Aim 3, the `druggability' of cinnamic aldehyde and structural analogs will be evaluated as tobacco cessation agents, ultimately in cryopreserved human hepatocytes. These compounds will also be evaluated for toxicity and pharmacokinetic factors, such as compound stability. This project represents the first known attempt to use the cinnamic aldehyde structure as a prototype from which to design novel smoking cessation agents, which is a long-term goal of this research. Describing the agents' pharmacokinetic characteristics is an essential step for translating selective in vitro inhibitors to viable drug candidates. Thus, if the results from this work support our hypothesis, it will lay the groundwork for future translational studies, which would evaluate the degree to which the agents inhibit nicotine metabolism in vivo, for example in mice, which also utilize CYP2A for nicotine metabolism. This work will also advance our knowledge of how aldehyde-containing agents inhibit cytochrome P450 enzymes, a large family of enzymes from which several isoforms are therapeutic targets. This R15 application offers excellent interdisciplinary research training opportunities for undergraduate and pharmacy students, and will significantly enhance the research infrastructure at Pacific University.